The long-term objective of this study is to reveal the structure and function relationships of the nuclear receptor peroxisome proliferator-activated receptor ? (PPAR?), which regulates a wide range of human physiology including adipocyte differentiation, glucose homeostasis, and inflammatory responses. PPAR? ligands such as rosiglitazone (AvandiaTM) and pioglitazone (ActosTM) are currently used in the treatment of type 2 diabetes in humans. In monocytes/macrophages, PPAR? is able to reduce the levels of cytokines (TNFa, interleukin-[unreadable], and interleukin-6) by inhibiting the expression of these pro-inflammatory genes. This anti-inflammatory effect of PPAR? is highly associated with the treatment of atherosclerosis. Therefore, PPAR? is an attractive therapeutic target for the treatment of such cardiovascular diseases. While many synthetic ligands have been developed, the physiological ligands of PPAR? remain a matter of great debate. The recently identified endogenous PPAR? ligands like nitrolinoleic acid (LNO2) are abundant in vivo and may help elucidate the physiological relevance of PPAR? and NO signaling in cardiovascular diseases. As a nuclear receptor, PPAR? mediates its function through ligand binding to its C-terminal ligand-binding domain (LBD) and targeted gene promoter binding to its middle DNA-binding domain (DBD). PPAR? also contains a potent N- terminal activation domain, AF1. Until recently, structures were available only for the isolated PPAR? LBD bound to diverse synthetic ligands and to short peptide motifs of coactivators. Clearly, this limited amount of structural information is a serious deficiency considering the importance of PPAR? in diabetes and cardiovascular diseases. In this study, we propose to fill in this knowledge gap by solving the crystal structures of various PPAR? complexes. Our specific aims are focused on the crystallization and structural determination of 1) the PPAR? LBD bound to LNO2, which will provide the first view of PPAR? bound to a natural ligand;2) the PPAR? LBD in complex with the PPAR? coactivator PGC1a;and 3) a PPAR? DBD-hinge-LBD fragment bound to RXR and a DNA of the PPAR response element (PPRE). The hypothesis for our specific aims is that the molecular interactions (protein-protein, protein-DNA, and protein-ligand) observed in those crystal structures will be crucial for understanding the biological functions of PPAR?. Following the structural determination, we will identify key structural elements by scrutinizing and analyzing the structures, and we will collaborate with Steve Kliewer (University of Texas Southwestern Medical Center), Eugene Chen (University of Michigan), and Bruce Freeman (University of Pittsburgh) on site-directed mutagenesis and cell-based transcriptional assays to validate the functional significance of the key features identified. Relevance: The structural information generated in this application will significantly enhance our understanding of the molecular mechanisms of PPAR? functions, and it should provide a rational template of drug discovery for the treatment of diabetes and heart diseases.